The Cyclization Mechanism of Cyclodextrin Glycosyltransferase (CGTase) as Revealed by a γ-Cyclodextrin-CGTase Complex at Å. Cyclodextrin glycosyltransferase is an important enzyme of cyclodextrin synthesis . This article mainly discusses the recent progress of the application of. INDUSTRIAL MICROBIOLOGY. Cyclodextrin glycosyltransferase from Bacillus licheniformis: optimization of production and its properties. Cyclodextrina.

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Cyclodextrin glycosyltransferase from Bacillus licheniformis: Cyclodextrina glycosyltransferase de Bacillus licheniformis: Cyclodextrin glycosyltransferase EC 2. An alkalophilic Bacillus strain, isolated from cassava peels, was identified as Bacillus licheniformis. CGTase production by this strain was better when potato starch was used as carbon source, followed by cassava starch and amylopectin. Glucose and amylose, on the other hand, acted as synthesis repressors.

When the cultivation was supplemented with sodium ions and had the pH adjusted between 6. This data is interesting because it contradicts the concept that alkalophilic microorganisms do not grow in this pH range. After ultrafiltration-centrifugation, one protein of This protein was identified in plates with starch and phenolphthalein.

Km and Vmax values were 1.

The Application of Cyclodextrin Glycosyltransferase in Biological Science | OMICS International

Ciclodextrina glicosiltransferase EC 2. The major types of CDs contain 6, 7 and 8 glucose molecules linked by a glycosidic bonds to form a ring and are named as a -CD, b -CD and g -CD, respectively.

CDs have the ability to encapsulate other cyclodrxtrin within their ringed structures. The ability of these unusual molecules to form inclusion complexes, which glycosylrtansferase change the physical and chemical properties of guest molecules, offers a variety of potential uses for food, cosmetic and pharmaceutical industries 3, According to Van Der Veen et al.

Furthermore, crystalline complexes that are the results of this process are stable, a characteristic that can provide many benefits A detailed history of glycosgltransferase development of cyclodextrins up to was reviewed by French 6.

The CGTases are known to be produced by various genera of the bacterial kingdom such as BacillusKlebsiellaPseudomonasBrevibacteriumMicrococus and Clostridium 9. Bacillus is the main genera, with various producing species 1,2,3,8,17, hlycosyltransferase, They are also known to catalyze four different transferase reactions: CGTase that can synthesize predominantly one type of CD has commercial importance since the separation of a particular form of CD is expensive and time-consuming.

The majority of the CGTases, especially from the alkaliphilic bacteria, convert starch into b -CD as the main product, but still in a mixture of CDs of different ratios The industrial production of CGTase was made attractive only when alkaliphilic Bacillus species were introduced as producing organism This paper reports the production optimization and some biochemical properties of a CGTase produced by a strain of Bacillus licheniformis isolated from cassava culture soil.

Strain and culture conditions. The isolation was made from a suspension of cassava peels through dilution method in plates of Petri with solid media of Horikoshi and Akiba The isolated microorganism was submitted to the microscopical analysis and presented morphology compatible with an aerobic positive Gram bacilli, presented positive catalase and motility, being characterized as pertaining to the Bacillus genera.

The microorganism was later characterized as Bacillus licheniformisaccording to the Bergey’s manual. It was also tested the nutrient broth NB Difco with potato starch as carbon source. Glycossyltransferase capacity of growth of the microorganism in presence of 2.


Qualitative analysis of CGTase. The enzyme CGTase produced in different cultivation conditions pH, carbon source and NaCl presence was acetone-concentrated and analyzed qualitatively with phenolphthalein glycosyptransferase 2 m L aliquots of this concentrated in Petri plates with solid medium at pH This methodology revealed the presence of CGTase activity in glycosyltdansferase culture filtrates through halo formation with good reproducibility. Determination of the specific activity of CGTase.

The cyclization activity was determined according to Makela Reaction samples were removed at 0, 10, 20 and 30 min. The amount of cyclodextrin was determined by the decrease of the absorption at nm, after the addition of 1.

For the calibration curve0. An unit of CGTase activity was defined as the amount of enzyme that produces 1 m mol of b -CD per minute, under glycosyltransferasw assay conditions. The specific activity was expressed in units of activity by milligram of protein. Protein concentration was estimated according to Hartree 12using bovine serum albumin as pattern. Determination of the optimum temperature.

Two fractions of the culture filtrate were taken for the determination of the optimal hydrolysis temperature.

The first fraction was concentrated with acetone 1: The second fraction was added of acetone 1: Ultra filtration-centrifugation and analysis by electrophoresis. Proteins retained by the membranes were recovered with Milli-Q water. These proteins and the filtrates were submitted to denaturing condition electrophoresis 15using kit Amersham Biosciences LMW as markers phosphorylase b The non-denaturing condition electrophoresis was carried out according to Davis 5 and it allowed the identification of CGTase activity in the gel, using two polyacrylamide identical gels, being one stained and the other put on a plate containing starch, agar and phenolphthalein pH Determination of the kinetic parameters.

The Km and Vmax values were estimated using the Eadie-Hofstee plot. The growth of the B. It was also tested the nutrient broth NB with potato starch Table 1. After the growth of the microorganism in different conditions, the results demonstrated, through formation of the colorless halos, that it remained active and did not lose the capacity to produce the CGTase activity. It is known that Bacillus alkalophilics requires initial pH 9.

However, this work verified that in culture medium added of 0. These data are similar to those reported from Tsai et al. Although amylose is a less complex substrate, it did not act as inductor of the CGTase production experiment 3.

The CGTase activity level was very small when the Horikoshi and Akiba 14 medium was substituted by the nutrient broth experiment 5being detected small halos in the plates with cassava starch and amylopectin. It is possible that glucose acts as repressor of the synthesis of CGTase, since the culture filtrate of experiment 7 did not lead to the halo formation in none of the plates.

Specific activities were determined using culture filtrates from experiments 1 to 8 and 13 to 16, using potato starch as substrate Table 2. This result agrees to the qualitative analysis previously described. These data indicate that glucose exerts repressor effect of the synthesis enzymatic. Similar results were reported by Gawande 11 with B. All the experiments were followed by reducing sugar determination using DNS, to make sure that we were not working with amylases. Identification of the CGTase in polyacrylamide gel.


After the migration in the polyacrylamide gel, the CGTase activity was identified through the halo formation next to front Fig.

The Application of Cyclodextrin Glycosyltransferase in Biological Science

Determination of the optimum hydrolysis temperature. For the determination of the optimum hydrolysis temperature of CGTase produced in potato starch medium, precipitates 1, 2 and 3 described in Method were used. The precipitate 1 was constituted of high and low molecular mass proteins plus carbohydrates; the precipitate 2 was constituted of high molecular mass proteins with low amount of carbohydrates; and the precipitate 3 was constituted of low molecular mass proteins with high molecular mass carbohydrates.

Another hypothesis is the presence of more than one enzyme with CGTase activity, as demonstrated in Fig. The CGTase produced by B. Two membranes were used, kDa and 30 kDa, and four protein fractions were obtained: The results also showed that the sample of MW superior to 30 kDa presented CGTase positive activity halo formationwhile the sample of MW inferior of 30 kDa did not present any activity.

Both samples glycoeyltransferase MW inferior and superior to kDa presented halo formation. These results suggest that CGTase presents MW near to kDa, since a small amount of enzyme was filtered and another part was retained in the kDa membrane after the centrifugation. Furthermore, the results showed that the sample of MW inferior to kDa presented only one protein band with enzymatic activity, showing the purification by this process.

Estimate of the apparent molecular weight.

Cyclodextrin glycosyltransferase

The apparent molecular weight of the CGTase purified was CGTase of the Bacillus sp. AL-6 the molecular mass was 74 kDa 7 and 67 kDa for Bacillus sp. Thermococcus sp CGTase presented an apparent molecular mass of 83 kDa 24 and one produced by Bacillus alkalophilic of Brazilian soil had molecular mass of Km and Vmax were estimated to be 1.

These results showed that the enzyme presented high affinity for the substrate, when compared with literature data. Gawande and Patkar 10 reported Km of 1.

These values are slightly inferior to those obtained in the present work. Martins and Hatti-Kaul 18 determined Km of The purification and characterization showed that the studied enzyme is really a CGTase because there was no amylolytic activity in the obtained culture filtrate.

Km value of 1. The method used for qualitative analysis is interesting, fast and glyycosyltransferase. Biosynthesis of cyclodextrin glucosyltransferase by immobilized Bacillusamyloliquefaciens in batch and continuos cultures.

Cyclodextrin glycosyltransferase – Wikipedia

Purification and properties of Bacillus coagulans cyclomaltodextrin glucanotransferase. Production, characterization, and application of cyclodextrins. Cyclomaltodextrin glucanotransferase from Bacillus circulans E Method and application to human serum proteins. The Schardinger dextrins, In: Advances in Carbohydrate Chemistry. Academic Press, New York,p.